The present invention is directed to thiol-protected pyrimidine nucleotide analogs which can be used, as one example, for syntheses of DNA and RNA by chemical or enzymatic methods. The subject analogs include reagents suitable for DNA or RNA synthesis via phosphoramidite, H-phosphonate or phosphotriester chemistry as well as reagents suitable for use by RNA and DNA polymerases, including thermostable polymerases employed by PCR or other nucleic acid amplification techniques. Methods of synthesizing the nucleotide analogs are also provided by the present invention. The nucleotide analogs of this invention can thus be incorporated into oligonucleotides or polynucleotides, deprotected, and then derivatized with a functional group.
Oligonucleotides with a variety of modifications have widespread utility for many purposes, such as stabilizing oligonucleotides to degradation, introducing reporter groups, allowing site-specific delivery of therapeutics, and introducing crosslinkers. Such modifications can occur as modified internucleotide phosphate linkages or analogs of such linkages, modified sugars or modified bases. Additionally, 5'- or 3'-end conjugates of the oligonucleotides represent another class of modified oligonucleotides. The present invention relates to base-modified nucleotide analogs with protected thiol groups; these analogs are intermediates for chemical or enzymatic synthesis of oligonucleotides and polynucleotides.
The synthesis of oligoribonucleotides and oligodeoxyribonucleotides (i.e. oligonucleotides) containing base-modified nucleotides at specific positions provides a powerful tool in the analysis of protein-nucleic acid or nucleic acid-nucleic acid interactions. These oligonucleotides have many other potential uses, such as the site-directed delivery of therapeutics, utility as anti-sense therapeutics, and utility as diagnostic probes. Nucleotide analogs can be introduced into nucleic acids either enzymatically, utilizing DNA and RNA polymerases, or chemically, utilizing manual or automated synthesis. Preparation of such oligonucleotides by automated synthesis utilizing, for example, phosphoramidite nucleotides allows for incorporation of a broad range of nucleotide analogs without the restraints for specific substrate conformation of the nucleotides that is imposed by most polymerases. Often, nucleotide analogs containing photoreactive crosslinking groups are introduced into oligonucleotides to probe protein-nucleic acid interactions via photocrosslinking (for a partial review, see Hanna, 1989, Methods Enzymol. 180:383-409; and Hanna, 1996, Methods Enzymol, 273 Chapter 31). Deoxyoligonucleotides containing 4-thiothymidine have been prepared and used for photochemical crosslinking of proteins directly to the nucleotide bases through the group (Nikiforov et al., 1992, Nucleic Acids Res. 20:1209-14). Similarly, oligonucleotides containing 3-(aminopropyl)-2'-deoxyuridine have been prepared and the amino group subsequently modified with fluorescent, photoactive or other reporter groups (Gibson et al., 1987, Nucleic Acids Res. 15:6455-66). However, the former thiodeoxynucleotide suffers the disadvantage that if it is modified with a thiol modifying reagent the normal Watson-Crick base of the nucleotide is drastically affected, making this and similar analogs generally unsuitable for use in enzymatic nucleic acid synthesis.
Other analogs involving modifications at the C5 position of deoxyuridine have also been previously reported. The thiol-containing analog, 5-thiocyanatodeoxyuridine phosphoramidite, provided a 5-mercaptodeoxyuridine moiety within the oligonucleotide following reduction of the thiocyanate (Bradley & Hanna, 1992). However, the thiocyanato moiety displayed variable stability during synthesis of both the nucleotide and the oligonucleotide and therefore this compound did not represent an ideal analog for incorporation of 5-thiol modified nucleotides into nucleic acids. The syntheses of a series of phosphoramidites containing alkylthiol tethers at the C5 position of deoxyuridine has been reported (Goodwin & Glick, 1993). The thiol groups in these analogs are attached to the ring by either a three, four, or five carbon chain. The presence of the carbon chains makes the minimal distance between the molecular probes and the oligonucleotide greater than that which can be achieved with our analog. In addition, these compounds represent alkyl thiol analogs which have a lower reactivity for modification of the thiol group than the 5-mercaptopyrimidine analog. This is due to an increase in acidity of 5-mercaptopyrimidines (pKa.about.5-5.6) over alkylthiol moieties (pKa.about.8-10). Phosphoramidites containing alkylthiol tethers at the N3 position of thymidine have also been prepared, but the position of this modification results in a disruption of the Watson-Crick base pairing. These analogs have been used mainly for preparing disulfide cross-links in DNA for studies involving stem loop and triple helical structures (Glick, 1991; Goodwin et al., 1994).
Several 5-modified deoxyuridine phosphoramidites are commercially available which contain functional groups (i.e. carboxylic acids or alkyl amines) for post-modification following incorporation into the oligonucleotide. Likewise, alkyl thiol ethers of 5-mercaptodeoxyuridine containing protected carboxylic acids and alkyl amines have also been described (Bergstrom et al., 1991). The protected functional groups in these analogs are not attached directly to the ring but are positioned at the end of carbon chains. These groups are not as easily modified as mercaptans: the functional groups formed during post-synthetic modification are limited, and an easily cleavable group is not available. Thiol-containing phosphoramidites for incorporating 4-thiothymidine (Clivio et al., 1992; Xu et al., 1992c), 4-thiodeoxyuridine (Clivio et al., 1992; Coleman & Kesicki, 1994), 2-thiothymidine (Connolly & Newman, 1989; Kuimelis & Nambiar, 1994), 6-thiodeoxyguanosine (Christopherson & Broom, 1991; Waters & Connolly, 1992; Xu et al., 1992b) and 6-thioinosine (Clivio et al., 1992a) have been reported. These analogs can occupy internal positions within an oligonucleotide and can serve as photochemical crosslinkers. However, they cannot be further modified without disrupting Watson-Crick base pairing, and therefore, as photocrosslinking probes, these analogs are only useful for evaluating interactions which occur directly with the nucleotide base. Modification with other molecular probes (e.g., fluorescent tags) would also disrupt Watson-Crick base pairing. In addition, the deprotection of oligonucleotides containing these thiol-modified nucleotides must be carefully monitored to prevent conversion of these analogs to the corresponding oxygen and nitrogen derivatives.
Described herein are nucleotide analogs which can be used for site-specific modification of DNA or RNA at internal and terminal positions within the DNA or RNA sequence, and after modification, for molecular probes which can be placed at variable distances from the DNA or RNA backbone.
The present invention provides novel base-protected nucleotide analogs, both ribonucleotides and deoxynucleotides, that contain masked thiol groups on the 5 position of pyrimidines, which is not involved in Watson-Crick base pairing. These analogs can be incorporated into oligonucleotides via automated synthesis and isolated with the thiol protecting group intact. After removal of the thiol protecting group many types of functional groups, such as photocrosslinking agents, fluorescent tags, radioisotopes, biotin, reporter molecules, spin labels (e.g., commercially available proxyl or tempo), chemiluminescent, antigenic or other functional groups, can be site-specifically attached by utilizing thiol-modifying reagents. This feature adds a level of specificity to the oligonucleotide modifications not present with the amino-tagged analogs previously described (Gibson et al, 1987), and enables examination of molecular interactions that are not directly at the nucleotide base by allowing functional groups to be placed at varying distances from the base or helix strand. Since these analogs have the functional group attached via the sulfur atom, some have the further advantage of being cleavable under conditions which will not degrade or modify the oligonucleotide.